100 results on '"D. Furling"'
Search Results
2. New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1)
- Author
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Thomas A. Cooper, H. Jaeger, D. Furutama, M. Siciliano, Giovanni Antonini, Geneviève Gourdon, S. Michalowski, E. Eddy, R. Krahe, John W. Day, S. E. Harris, J. P. Barbet, M. Shimizu, G. B. Browne, M. Gosling, A. V. Philips, Loreto Martorell, P. Maire, Glenn E. Morris, Zeljka Korade, N. Carey, Richard R. Sinden, C. A. Thornton, A. M. Mitchell, M. Baiget, A. Balasubramanyam, L. P.W. Ranum, Shigeru Sato, M. Eriksson, T. Kobayashi, M. Khajavi, J. Mathieu, F. K. Gould, B. Eymard, D. Pribnow, R. H. Singer, J. D. Griffith, C. Liquori, M. Wagner, T. Ansved, D. E. Housman, N. Spring, A. Johansson, S. Salvatori, B. Luciano, Claudia Abbruzzese, I. Gonzales, J. Adelman, J. P. Mounsey, B. Wieringa, J. Waring, B. Perryman, D. Furling, M. Devillers, H. Furuya, F. Lehmann, H. Yamagata, M. S. Mahadevan, Darren G. Monckton, Geoffrey P. Miller, D. Hilton Jones, A. S. Lia-Baldini, J. Westerlaken, M. Swanson, S. J. Tapscott, T. R. Klesert, R. D. Wells, N. Ohsawa, H. Seznec, H. Moore, E. J. Chen, M. Hamshere, Tetsuo Ashizawa, U. Kvist, A. D. Roses, C. Junien, Catherine L Winchester, M. Gennarelli, M. Kinoshita, K. Johnson, Christopher E. Pearson, Lubov Timchenko, and J. R. Moorman
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musculoskeletal diseases ,Genetics ,Pathology ,medicine.medical_specialty ,Genetic heterogeneity ,Myotonic Disorder ,Locus (genetics) ,Biology ,medicine.disease ,Myotonia ,Myotonic dystrophy ,Proximal myotonic myopathy ,Atrophy ,medicine ,Neurology (clinical) ,Trinucleotide repeat expansion - Abstract
Myotonic dystrophy (DM; OMIM 160900, also known as dystrophia myotonica, myotonia atrophica and Steinert disease) is an autosomal dominant myotonic myopathy associated with abnormalities of other organs, including eyes, heart, endocrine system, central and peripheral nervous systems, gastrointestinal organs, bone, and skin.1 The mutation underlying DM is an expansion of an unstable cytosine-thymine-guanine (CTG) trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene in chromosome 19q13.3.2-4 In 1994, Thornton et al.5 described an autosomal dominant disorder similar to DM without CTG repeat expansion at the DM locus. Ricker et al.6 named this disease "proximal myotonic myopathy" (PROMM; OMIM 600109) because of predominantly proximal muscle weakness without atrophy as opposed to the distal muscle involvement seen in DM. Subsequently, Meola et al.7 described a variant of PROMM with unusual myotonic and myopathic features, which they named "proximal myotonic myopathy syndrome," and Udd et al.8 described a PROMM-like family with dystrophic features, which they named "proximal myotonic dystrophy" (PDM). Researchers at the University of Minnesota9,10 found another multisystemic myotonic disorder that closely resembles DM with distal muscle weakness but no CTG repeat expansion. Because of the close phenotypic resemblance to DM, they called this disease "myotonic dystrophy type 2" (DM2; OMIM 602668). In 1998, Ranum et al.9 assigned the DM2 locus to chromosome 3q in a large kindred. Shortly after that, Ricker et al.11 found that the majority of German PROMM families show linkage to the DM2 locus. PDM was also mapped to this region (Krahe and Udd, personal communication, 1999). Whether PROMM, PDM, and DM2 represent different phenotypic expressions of a disease caused by the same mutation or if they are allelic disorders remains to be determined. It is also possible that these disorders are caused by mutations in different genes that are closely linked in the chromosome 3q region.12 Furthermore, the disease loci in some typical PROMM families11 and other families with multisystemic myotonic disorders have been excluded from both DM and DM2 loci. Because of the genetic and phenotypic heterogeneity in this group of disorders, it became necessary to establish a new nomenclature foreseeing the future discovery of new disease loci and phenotypic variability.
- Published
- 2000
3. Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1
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L-E, Thornell, M, Lindstöm, V, Renault, A, Klein, V, Mouly, T, Ansved, G, Butler-Browne, and D, Furling
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Adult ,Male ,Satellite Cells, Skeletal Muscle ,Cell Count ,Middle Aged ,Protein Serine-Threonine Kinases ,Telomere ,Immunohistochemistry ,Severity of Illness Index ,Myotonin-Protein Kinase ,Muscular Atrophy ,Young Adult ,Humans ,Myotonic Dystrophy ,Regeneration ,Female ,Muscle, Skeletal ,Trinucleotide Repeat Expansion ,Cells, Cultured ,Cell Proliferation - Abstract
Myotonic dystrophy type 1 (DM1), one of the most common forms of inherited neuromuscular disorders in the adult, is characterized by progressive muscle weakness and wasting leading to distal muscle atrophy whereas proximal muscles of the same patients are spared during the early phase of the disease. In this report, the role of satellite cell dysfunction in the progressive muscular atrophy has been investigated.Biopsies were obtained from distal and proximal muscles of the same DM1 patients. Histological and immunohistological analyses were carried out and the past regenerative history of the muscle was evaluated. Satellite cell number was quantified in vivo and proliferative capacity was determined in vitro.The size of the CTG expansion was positively correlated with the severity of the symptoms and the degree of muscle histopathology. Marked atrophy associated with typical DM1 features was observed in distal muscles of severely affected patients whereas proximal muscles were relatively spared. The number of satellite cells was significantly increased (twofold) in the distal muscles whereas very little regeneration was observed as confirmed by telomere analyses and developmental MyHC staining (0.3-3%). The satellite cells isolated from the DM1 distal muscles had a reduced proliferative capacity (36%) and stopped growing prematurely with telomeres longer than control cells (8.4 vs. 7.1 kb), indicating that the behaviour of these precursor cells was modified.Our results indicate that alterations in the basic functions of the satellite cells progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.
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- 2009
4. Basic transcription element-binding protein (BTEB) is a thyroid hormone-regulated gene in the developing central nervous system. Evidence for a role in neurite outgrowth
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R J, Denver, L, Ouellet, D, Furling, A, Kobayashi, Y, Fujii-Kuriyama, and J, Puymirat
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Neurons ,Kruppel-Like Transcription Factors ,Brain ,Gene Expression Regulation, Developmental ,Rats ,Up-Regulation ,DNA-Binding Proteins ,Rats, Sprague-Dawley ,Pregnancy ,Astrocytes ,Neurites ,Animals ,Triiodothyronine ,Female ,RNA, Messenger ,Cell Division ,Cells, Cultured ,Transcription Factors - Abstract
Thyroid hormone (3,5,3'-triiodothyronine; T(3)) is essential for normal development of the vertebrate brain, influencing diverse processes such as neuronal migration, myelin formation, axonal maturation, and dendritic outgrowth. We have identified basic transcription element-binding protein (BTEB), a small GC box-binding protein, as a T(3)-regulated gene in developing rat brain. BTEB mRNA levels in cerebral cortex exhibit developmental regulation and thyroid hormone dependence. T(3) regulation of BTEB mRNA is neural cell-specific, being up-regulated in primary cultures of embryonic neurons (E16) and in neonatal astrocytes (P2), but not in neonatal oligodendrocytes (P2). T(3) rapidly up-regulated BTEB mRNA in neuro-2a cells engineered to express thyroid hormone receptor (TR) beta1 but not in cells expressing TRalpha1, suggesting that the regulation of this gene is specific to the TRbeta1 isoform. Several lines of evidence support a transcriptional action of T(3) on BTEB gene expression. Overexpression of BTEB in Neuro-2a cells dramatically increased the number and length of neurites in a dose-dependent manner suggesting a role for this transcription factor in neuronal process formation. However, other T(3)-dependent changes were not altered; i.e. overexpression of BTEB had no effect on the rate of cell proliferation nor on the expression of acetylcholinesterase activity.
- Published
- 1999
5. P81 Compound screening in myotonic dystrophy
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X. Li, I. Udosen, Christopher J. Hayes, T.E. Robinson, Ami Ketley, D. Furling, C. Austin, S. Arya, C.Z. Chen, and J.D. Brook
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Pathology ,medicine.medical_specialty ,Neurology ,business.industry ,Pediatrics, Perinatology and Child Health ,Medicine ,Neurology (clinical) ,business ,medicine.disease ,Myotonic dystrophy ,Genetics (clinical) - Published
- 2012
6. Glutathione peroxidase-mediated inhibition of DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma cells
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Marc-Edouard Mirault, P Petrov, G Poirier, and D Furling
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chemistry.chemical_classification ,Neuroblastoma cell ,Hydroxydopamine ,chemistry ,DNA damage ,Apoptosis ,Glutathione peroxidase ,Cell Biology ,Molecular Biology ,Biochemistry ,Molecular biology - Published
- 1997
7. Extended Amplification In Vitro and Replicative Senescence: Key Factors Implicated in the Success of Human Myoblast Transplantation.
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R.N. Cooper, D. Thiesson, D. Furling, J.P. Di Santo, G.S. Butler-Browne, and V. Mouly
- Published
- 2003
8. Treadmill running and mechanical overloading improved the strength of the plantaris muscle in the dystrophin-desmin double knockout (DKO) mouse.
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Moutachi D, Hyzewicz J, Roy P, Lemaitre M, Bachasson D, Amthor H, Ritvos O, Li Z, Furling D, Agbulut O, and Ferry A
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- Animals, Male, Mice, Mice, Inbred C57BL, Mice, Inbred mdx, Mice, Knockout, Muscle Contraction, Muscle Strength, Muscular Dystrophy, Duchenne genetics, Muscular Dystrophy, Duchenne physiopathology, Desmin genetics, Desmin metabolism, Dystrophin genetics, Muscle, Skeletal physiology, Muscle, Skeletal metabolism, Physical Conditioning, Animal, Running physiology
- Abstract
Limited knowledge exists regarding the chronic effect of muscular exercise on muscle function in a murine model of severe Duchenne muscular dystrophy (DMD). Here we determined the effects of 1 month of voluntary wheel running (WR), 1 month of enforced treadmill running (TR) and 1 month of mechanical overloading resulting from the removal of the synergic muscles (OVL) in mice lacking both dystrophin and desmin (DKO). Additionally, we examined the effect of activin receptor administration (AR). DKO mice, displaying severe muscle weakness, atrophy and greater susceptibility to contraction-induced functional loss, were exercised or treated with AR at 1 month of age and in situ force production of lower leg muscle was measured at the age of 2 months. We found that TR and OVL increased absolute maximal force and the rate of force development of the plantaris muscle in DKO mice. In contrast, those of the tibialis anterior (TA) muscle remained unaffected by TR and WR. Furthermore, the effects of TR and OVL on plantaris muscle function in DKO mice closely resembled those in mdx mice, a less severe murine DMD model. AR also improved absolute maximal force and the rate of force development of the TA muscle in DKO mice. In conclusion, exercise training improved plantaris muscle weakness in severely affected dystrophic mice. Consequently, these preclinical results may contribute to fostering further investigations aimed at assessing the potential benefits of exercise for DMD patients, particularly resistance training involving a low number of intense muscle contractions. KEY POINTS: Very little is known about the effects of exercise training in a murine model of severe Duchenne muscular dystrophy (DMD). One reason is that it is feared that chronic muscular exercise, particularly that involving intense muscle contractions, could exacerbate the disease. In DKO mice lacking both dystrophin and desmin, characterized by severe lower leg muscle weakness, atrophy and fragility in comparison to the less severe DMD mdx model, we found that enforced treadmill running improved absolute maximal force of the plantaris muscle, while that of tibialis anterior muscle remained unaffected by both enforced treadmill and voluntary wheel running. Furthermore, mechanical overloading, a non-physiological model of chronic resistance exercise, reversed plantaris muscle weakness. Consequently, our findings may have the potential to alleviate concerns and pave the way for exploring the prescription of endurance and resistance training as a viable therapeutic approach for the treatment of dystrophic patients. Additionally, such interventions may serve in mitigating the pathophysiological mechanisms induced by physical inactivity., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)
- Published
- 2024
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9. Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy.
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Hassani M, Moutachi D, Lemaitre M, Boulinguiez A, Furling D, Agbulut O, and Ferry A
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- Humans, Animals, Mice, Mice, Inbred mdx, Calcineurin metabolism, Quality of Life, Muscle, Skeletal metabolism, Disease Models, Animal, Muscular Dystrophy, Duchenne pathology, Resistance Training, Cyclosporins pharmacology
- Abstract
Mechanical overloading (OVL) resulting from the ablation of muscle agonists, a supra-physiological model of resistance training, reduces skeletal muscle fragility, i.e. the immediate maximal force drop following lengthening contractions, and increases maximal force production, in mdx mice, a murine model of Duchene muscular dystrophy (DMD). Here, we further analyzed these beneficial effects of OVL by determining whether they were blocked by cyclosporin, an inhibitor of the calcineurin pathway, and whether there were also observed in the D2-mdx mice, a more severe murine DMD model. We found that cyclosporin did not block the beneficial effect of 1-month OVL on plantaris muscle fragility in mdx mice, nor did it limit the increases in maximal force and muscle weight (an index of hypertrophy). Fragility and maximal force were also ameliorated by OVL in the plantaris muscle of D2-mdx mice. In addition, OVL increased the expression of utrophin, cytoplamic γ-actin, MyoD, and p-Akt in the D2-mdx mice, proteins playing an important role in fragility, maximal force gain and muscle growth. In conclusion, OVL reduced fragility and increased maximal force in the more frequently used mild mdx model but also in D2-mdx mice, a severe model of DMD, closer to human physiopathology. Moreover, these beneficial effects of OVL did not seem to be related to the activation of the calcineurin pathway. Thus, this preclinical study suggests that resistance training could have a potential benefit in the improvement of the quality of life of DMD patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hassani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2024
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10. Valproic acid reduces muscle susceptibility to contraction-induced functional loss but increases weakness in two murine models of Duchenne muscular dystrophy.
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Moutachi D, Lemaitre M, Delacroix C, Agbulut O, Furling D, and Ferry A
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- Female, Animals, Mice, Valproic Acid pharmacology, Valproic Acid metabolism, Mice, Inbred mdx, Utrophin metabolism, Disease Models, Animal, Desmin metabolism, Muscle Contraction physiology, Muscle, Skeletal metabolism, Muscular Dystrophy, Duchenne drug therapy, Muscular Dystrophy, Duchenne metabolism, Muscular Dystrophy, Duchenne pathology
- Abstract
Skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) are more susceptible to contraction-induced functional loss, which is not related to fatigue. Valproic acid (VPA) reportedly improves serological and histological markers of damage in dystrophin-deficient murine muscle. Here, we tested whether VPA would reduce the susceptibility to contraction-induced functional loss in two murine DMD models. Adult female mdx (mild) and D2-mdx (severe) DMD murine models were administered VPA (240 mg/kg) or saline for 7 days. Some VPA-treated mdx mice also performed voluntary running in a wheel, which is known to reduce the susceptibility to contraction-induced functional loss; that is, isometric force drop following eccentric contractions. In situ muscle function was assessed before, during and after eccentric contractions. Muscle utrophin and desmin expression were also evaluated using immunoblotting. Interestingly, VPA reduced the isometric force drop following eccentric contractions in both murine models, without change in the relative eccentric maximal force and in the expression of utrophin and desmin. VPA for 7 days combined with voluntary running had no additive effect compared to VPA alone. Furthermore, VPA reduced the absolute isometric maximal force before eccentric contractions in both murine models. The results of our study indicated that VPA in both murine DMD models reduced the susceptibility to contraction-induced functional loss but increased muscle weakness., (© 2023 The Authors. Clinical and Experimental Pharmacology and Physiology published by John Wiley & Sons Australia, Ltd.)
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- 2023
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11. Specific DMPK -promoter targeting by CRISPRi reverses myotonic dystrophy type 1-associated defects in patient muscle cells.
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Porquet F, Weidong L, Jehasse K, Gazon H, Kondili M, Blacher S, Massotte L, Di Valentin E, Furling D, Gillet NA, Klein AF, Seutin V, and Willems L
- Abstract
Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats ( CUGexp ). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK -promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp -RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK -promoter silencing by CRISPRi as a promising therapeutic approach for DM1., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)
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- 2023
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12. MBNL-dependent impaired development within the neuromuscular system in myotonic dystrophy type 1.
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Tahraoui-Bories J, Mérien A, González-Barriga A, Lainé J, Leteur C, Polvèche H, Carteron A, De Lamotte JD, Nicoleau C, Polentes J, Jarrige M, Gomes-Pereira M, Ventre E, Poydenot P, Furling D, Schaeffer L, Legay C, and Martinat C
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- Adult, Humans, RNA-Binding Proteins metabolism, Neuromuscular Junction pathology, Motor Neurons pathology, Myotonic Dystrophy pathology, Induced Pluripotent Stem Cells metabolism
- Abstract
Aims: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1., Methods: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells., Results: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1., Conclusions: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays., (© 2022 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)
- Published
- 2023
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13. Dystrophin Restoration after Adeno-Associated Virus U7-Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model.
- Author
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Monceau A, Moutachi D, Lemaitre M, Garcia L, Trollet C, Furling D, Klein A, and Ferry A
- Abstract
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by Dmd mutations, resulting in the absence of dystrophin in skeletal muscle, and a greater susceptibility to damage during contraction (exercise). The current study evaluated whether voluntary exercise impacts a Dmd exon skipping and muscle physiology in a severe DMD murine model. D2-mdx mice were intramuscularly injected with an adeno-associated virus (AAV) U7 snRNA to correct Dmd reading frame, and allowed to voluntary run on a wheel for 1 month. Voluntary running did not induce muscle fiber regeneration, as indicated by the percentage of centronucleated fibers, Myh3 and Myh4 expression, and maximal force production, and thus possibly did not compromise the gene therapy approach. Voluntary running did not impact the number of viral genomes and the expression of U7 and Dmd 1 month after injection of AAV-U7 injected just before exercise initiation, but reduced the amount of dystrophin in dystrophin-expressing fibers from 80% to 65% of the muscle cross-sectional area. In conclusion, voluntary running did not induce muscle damage and had no drastic detrimental effect on the AAV gene therapy exon skipping approach in a severe murine DMD model. Moreover, these results suggest considering exercise as an additional element in the design and conception of future therapeutic approaches for DMD., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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14. Identification of a CCG-Enriched Expanded Allele in Patients with Myotonic Dystrophy Type 1 Using Amplification-Free Long-Read Sequencing.
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Tsai YC, de Pontual L, Heiner C, Stojkovic T, Furling D, Bassez G, Gourdon G, and Tomé S
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- Humans, Myotonin-Protein Kinase genetics, Alleles, Trinucleotide Repeat Expansion genetics, Genetic Counseling, Myotonic Dystrophy diagnosis, Myotonic Dystrophy genetics
- Abstract
Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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15. The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle.
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Monceau A, Delacroix C, Lemaitre M, Revet G, Furling D, Agbulut O, Klein A, and Ferry A
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- Animals, Genetic Therapy, Mice, Mice, Inbred C57BL, Mice, Inbred mdx, Muscle Contraction physiology, Muscle, Skeletal physiology, Muscular Dystrophy, Animal genetics, Muscular Dystrophy, Animal metabolism, Muscular Dystrophy, Animal therapy, Muscular Dystrophy, Duchenne genetics, Muscular Dystrophy, Duchenne metabolism, Muscular Dystrophy, Duchenne therapy
- Abstract
Purpose: Greater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle damage, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) using an AAV on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle., Methods: Both tibialis anterior muscles of the same mdx mouse received the transfer of Prox1 and PBS and the mice performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular muscle parameters were also evaluated., Results: Interestingly, Prox1 transfer reduced the isometric force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the absolute maximal isometric force (p < 0.01), but not the specific maximal isometric force, before lengthening contraction in exercised (p < 0.01) and sedentary mdx mice., Conclusion: Our results indicate that Prox1 transfer increased the beneficial effect of chronic exercise on muscle fragility in mdx mice, but reduced absolute maximal force. Thus, the potential clinical benefit of the transfer of Prox1 into exercised dystrophic muscle can merit further investigation., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
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16. Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats.
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Arandel L, Matloka M, Klein AF, Rau F, Sureau A, Ney M, Cordier A, Kondili M, Polay-Espinoza M, Naouar N, Ferry A, Lemaitre M, Begard S, Colin M, Lamarre C, Tran H, Buée L, Marie J, Sergeant N, and Furling D
- Subjects
- Animals, Cell Nucleus genetics, Cell Nucleus metabolism, Cell Nucleus pathology, Humans, Mice, Muscle, Skeletal metabolism, RNA genetics, RNA metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Myotonic Dystrophy genetics, Myotonic Dystrophy metabolism, Myotonic Dystrophy therapy
- Abstract
Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals' skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2022
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17. Comprehensive transcriptome-wide analysis of spliceopathy correction of myotonic dystrophy using CRISPR-Cas9 in iPSCs-derived cardiomyocytes.
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Dastidar S, Majumdar D, Tipanee J, Singh K, Klein AF, Furling D, Chuah MK, and VandenDriessche T
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- Alternative Splicing, CRISPR-Cas Systems, Calmodulin-Binding Proteins genetics, Humans, Myocytes, Cardiac metabolism, Myotonin-Protein Kinase genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Transcriptome, Trinucleotide Repeat Expansion genetics, Induced Pluripotent Stem Cells metabolism, Myotonic Dystrophy genetics, Myotonic Dystrophy therapy
- Abstract
CTG repeat expansion (CTG
exp ) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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18. CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis.
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Mérien A, Tahraoui-Bories J, Cailleret M, Dupont JB, Leteur C, Polentes J, Carteron A, Polvèche H, Concordet JP, Pinset C, Jarrige M, Furling D, and Martinat C
- Subjects
- Alternative Splicing, Gene Editing, Humans, Muscle Development genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Induced Pluripotent Stem Cells metabolism, Myotonic Dystrophy pathology
- Abstract
Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2021
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19. [How alternative splicing contributes to clathrin's structural plasticity].
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Moulay G, Bitoun M, Furling D, and Vassilopoulos S
- Subjects
- Humans, Alternative Splicing, Clathrin chemistry, Clathrin genetics, Clathrin metabolism
- Published
- 2021
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20. Cas9 targeting of toxic foci of RNA repeats.
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Furling D
- Subjects
- Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Mice, Phenotype, RNA genetics, CRISPR-Cas Systems genetics, Myotonic Dystrophy genetics
- Published
- 2021
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21. A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1.
- Author
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Zhang N, Bewick B, Xia G, Furling D, and Ashizawa T
- Abstract
Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii ( Lsh ) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) - a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that Lsh Cas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, Lsh Cas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated Lsh Cas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of Lsh Cas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Zhang, Bewick, Xia, Furling and Ashizawa.)
- Published
- 2020
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22. New recessive mutations in SYT2 causing severe presynaptic congenital myasthenic syndromes.
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Bauché S, Sureau A, Sternberg D, Rendu J, Buon C, Messéant J, Boëx M, Furling D, Fauré J, Latypova X, Gelot AB, Mayer M, Mary P, Whalen S, Fournier E, Cloix I, Remerand G, Laffargue F, Nougues MC, Fontaine B, Eymard B, Isapof A, and Strochlic L
- Abstract
Objective: To report the identification of 2 new homozygous recessive mutations in the synaptotagmin 2 ( SYT2 ) gene as the genetic cause of severe and early presynaptic forms of congenital myasthenic syndromes (CMSs)., Methods: Next-generation sequencing identified new homozygous intronic and frameshift mutations in the SYT2 gene as a likely cause of presynaptic CMS. We describe the clinical and electromyographic patient phenotypes, perform ex vivo splicing analyses to characterize the effect of the intronic mutation on exon splicing, and analyze the functional impact of this variation at the neuromuscular junction (NMJ)., Results: The 2 infants presented a similar clinical phenotype evoking first a congenital myopathy characterized by muscle weakness and hypotonia. Next-generation sequencing allowed to the identification of 1 homozygous intronic mutation c.465+1G>A in patient 1 and another homozygous frameshift mutation c.328_331dup in patient 2, located respectively in the 5' splice donor site of SYT2 intron 4 and in exon 3. Functional studies of the intronic mutation validated the abolition of the splice donor site of exon 4 leading to its skipping. In-frame skipping of exon 4 that encodes part of the C2A calcium-binding domain of SYT2 is associated with a loss-of-function effect resulting in a decrease of neurotransmitter release and severe pre- and postsynaptic NMJ defects., Conclusions: This study identifies new homozygous recessive SYT2 mutations as the underlying cause of severe and early presynaptic form of CMS expanding the genetic spectrum of recessive SYT2 -related CMS associated with defects in neurotransmitter release., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)
- Published
- 2020
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23. Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques.
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Moulay G, Lainé J, Lemaître M, Nakamori M, Nishino I, Caillol G, Mamchaoui K, Julien L, Dingli F, Loew D, Bitoun M, Leterrier C, Furling D, and Vassilopoulos S
- Subjects
- Adult, Animals, Cell Membrane metabolism, Child, Endocytosis physiology, Exons physiology, Female, Humans, Male, Mice, Mice, Inbred C57BL, Muscle Fibers, Skeletal metabolism, Young Adult, Alternative Splicing physiology, Clathrin metabolism, Clathrin Heavy Chains metabolism, Coated Pits, Cell-Membrane metabolism
- Abstract
Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance., (© 2020 Moulay et al.)
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- 2020
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24. Desmin prevents muscle wasting, exaggerated weakness and fragility, and fatigue in dystrophic mdx mouse.
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Ferry A, Messéant J, Parlakian A, Lemaitre M, Roy P, Delacroix C, Lilienbaum A, Hovhannisyan Y, Furling D, Klein A, Li Z, and Agbulut O
- Subjects
- Animals, Desmin genetics, Disease Models, Animal, Dystrophin genetics, Mice, Mice, Inbred mdx, Muscle, Skeletal, Muscular Dystrophy, Duchenne genetics
- Abstract
Key Points: Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix. Deletion of the desmin gene in mdx mice [double knockout (DKO) mice] induces marked muscle weakness and fatigue resistance compared to mdx mice. Muscle fragility (higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice. By contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy. Desmin cDNA transfer with adeno-associated virus in newborn mdx mice reduced muscle weakness. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic muscle., Abstract: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by dystrophin deficiency. Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix that contributes to muscle function. In the present study, we attempted to provide further insight into the roles of desmin, for which the expression is increased in the muscle from the mouse mdx DMD model. We show that a deletion of the desmin gene (Des) in mdx mice [double knockout (DKO) mice, mdx:desmin-/-] induces a marked muscle weakness; namely, a reduced absolute maximal force production and increased fatigue compared to that in mdx mice. Fragility (i.e. higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice, despite the promotion of supposedly less fragile muscle fibres in DKO mice, and this worsening of fragility was related to a decreased muscle excitability. Moreover, in contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy, as indicated by smaller and fewer fibres, with a reduced percentage of centronucleated fibres, potentially explaining the severe muscle weakness. Notably, Desmin cDNA transfer with adeno-associated virus in newborn mdx mice improved specific maximal force normalized to muscle weight. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic mdx mice, which differ, at least in part, from those observed in healthy muscle., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)
- Published
- 2020
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25. Cardiovascular manifestations of myotonic dystrophy.
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Wahbi K and Furling D
- Subjects
- Animals, Arrhythmias, Cardiac genetics, Arrhythmias, Cardiac mortality, Arrhythmias, Cardiac physiopathology, Death, Sudden, Cardiac epidemiology, Defibrillators, Implantable, Genetic Predisposition to Disease, Humans, Myotonic Dystrophy genetics, Myotonic Dystrophy mortality, Myotonic Dystrophy physiopathology, Pacemaker, Artificial, Prevalence, Risk Factors, Treatment Outcome, Arrhythmias, Cardiac therapy, Cardiac Pacing, Artificial adverse effects, Cardiac Pacing, Artificial mortality, Death, Sudden, Cardiac prevention & control, Electric Countershock adverse effects, Electric Countershock instrumentation, Electric Countershock mortality, Myotonic Dystrophy epidemiology
- Abstract
Patients with myotonic dystrophy, the most common neuromuscular dystrophy in adults, have a high prevalence of arrhythmic complications with increased cardiovascular mortality and high risk for sudden death. Sudden death prevention is central and relies on annual follow-up and prophylactic permanent pacing in patients with conduction defects on electrocardiogram and/or infrahisian blocks on electrophysiological study. Implantable cardiac defibrillator therapy may be indicated in patients with ventricular tachyarrhythmia., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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26. miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy.
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Sabater-Arcis M, Bargiela A, Furling D, and Artero R
- Abstract
Unstable CTG expansions in the 3' UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy- and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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27. FISH Protocol for Myotonic Dystrophy Type 1 Cells.
- Author
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Klein AF, Arandel L, Marie J, and Furling D
- Subjects
- Cell Nucleus genetics, Cells, Cultured, Fluorescent Antibody Technique, Humans, Mutation, Single Molecule Imaging, Trinucleotide Repeat Expansion, In Situ Hybridization, Fluorescence methods, Myotonic Dystrophy genetics, Myotonin-Protein Kinase genetics, RNA-Binding Proteins genetics
- Abstract
Mutant DMPK transcripts containing expanded CUG repeats (CUGexp) are retained within the nucleus of myotonic dystrophy type 1 (DM1) cells as discrete foci. Nuclear CUGexp-RNA foci that sequester MBNL1 splicing factor represent a hallmark of this RNA dominant disease caused by the expression of expanded microsatellite repeats. Here we described fluorescent in situ hybridization (FISH) techniques to detect either RNA containing CUG expansion or DMPK transcripts in human DM1 or WT cells. In addition, we propose a combined FISH/immunofluorescence protocol to visualize the colocalization of MBNL1 with CUGexp-RNA foci in DM1 cells.
- Published
- 2020
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28. Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.
- Author
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Klein AF, Varela MA, Arandel L, Holland A, Naouar N, Arzumanov A, Seoane D, Revillod L, Bassez G, Ferry A, Jauvin D, Gourdon G, Puymirat J, Gait MJ, Furling D, and Wood MJ
- Subjects
- Animals, Cells, Cultured, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Dose-Response Relationship, Drug, Humans, Mice, Muscle, Skeletal pathology, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Cell-Penetrating Peptides pharmacology, Muscle, Skeletal metabolism, Myotonic Dystrophy drug therapy, Myotonic Dystrophy genetics, Myotonic Dystrophy metabolism, Myotonic Dystrophy pathology, Myotonin-Protein Kinase genetics, Myotonin-Protein Kinase metabolism, Oligodeoxyribonucleotides, Antisense genetics, Oligodeoxyribonucleotides, Antisense pharmacology
- Abstract
Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.
- Published
- 2019
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29. Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.
- Author
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Lo Scrudato M, Poulard K, Sourd C, Tomé S, Klein AF, Corre G, Huguet A, Furling D, Gourdon G, and Buj-Bello A
- Subjects
- Alternative Splicing, Animals, Base Sequence, CRISPR-Cas Systems, Cell Nucleus, Disease Models, Animal, Fluorescent Antibody Technique, Gene Expression, Gene Targeting, Genetic Vectors genetics, Humans, Mice, Mice, Knockout, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Myotonic Dystrophy genetics, Myotonic Dystrophy therapy, RNA, Guide, CRISPR-Cas Systems, Transduction, Genetic, Gene Editing, Myotonin-Protein Kinase genetics, RNA, Nuclear, Trinucleotide Repeat Expansion
- Abstract
Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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30. Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.
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Voellenkle C, Perfetti A, Carrara M, Fuschi P, Renna LV, Longo M, Sain SB, Cardani R, Valaperta R, Silvestri G, Legnini I, Bozzoni I, Furling D, Gaetano C, Falcone G, Meola G, and Martelli F
- Subjects
- Adult, Alternative Splicing genetics, Case-Control Studies, Cell Line, Female, Humans, Male, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Myotonic Dystrophy blood, Polymerase Chain Reaction, RNA blood, RNA, Circular, Reproducibility of Results, Gene Expression Regulation, Myotonic Dystrophy genetics, RNA genetics
- Abstract
Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by "back-splicing", which is the linking covalently of 3'- and 5'-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.
- Published
- 2019
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31. Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1.
- Author
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Maury Y, Poydenot P, Brinon B, Lesueur L, Gide J, Roquevière S, Côme J, Polvèche H, Auboeuf D, Alexandre Denis J, Pietu G, Furling D, Lechuga M, Baghdoyan S, Peschanski M, and Martinat C
- Abstract
There is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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32. Improvement of Dystrophic Muscle Fragility by Short-Term Voluntary Exercise through Activation of Calcineurin Pathway in mdx Mice.
- Author
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Delacroix C, Hyzewicz J, Lemaitre M, Friguet B, Li Z, Klein A, Furling D, Agbulut O, and Ferry A
- Subjects
- Animals, Mice, Mice, Inbred mdx, Motor Activity, Muscle Contraction, Muscular Dystrophy, Animal metabolism, Muscular Dystrophy, Animal pathology, Calcineurin metabolism, Muscular Dystrophy, Animal prevention & control, Physical Conditioning, Animal
- Abstract
Dystrophin deficiency in mdx mice, a model for Duchenne muscular dystrophy, leads to muscle weakness revealed by a reduced specific maximal force as well as fragility (ie, higher susceptibility to contraction-induced injury, as shown by a greater force decrease after lengthening contractions). Both symptoms could be improved with dystrophin restoration-based therapies and long-term (months) voluntary exercise. Herein, we evaluated the effect of short-term (1-week) voluntary wheel running. We found that running improved fragility of tibialis anterior muscle (TA), but not plantaris muscle, independently of utrophin up-regulation, without affecting weakness. Moreover, TA muscle excitability was also preserved by running, as shown by compound muscle action potential measurements after lengthening contractions. Of interest, the calcineurin inhibitor cyclosporin A prevented the effect of running on both muscle fragility and excitability. Cyclosporin also prevented the running-induced changes in expression of genes involved in excitability (Scn4a and Cacna1s) and slower contractile phenotype (Myh2 and Tnni1) in TA muscle. In conclusion, short-term voluntary exercise improves TA muscle fragility in mdx mice, without worsening weakness. Its effect was related to preserved excitability, calcineurin pathway activation, and changes in the program of genes involved in excitability and slower contractile phenotype. Thus, remediation of muscle fragility of Duchenne muscular dystrophy patients through appropriate exercise training deserves to be explored in more detail., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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33. Activation of the interferon type I response rather than autophagy contributes to myogenesis inhibition in congenital DM1 myoblasts.
- Author
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Rizzo M, Beffy P, Del Carratore R, Falleni A, Pretini V, D'Aurizio R, Botta A, Evangelista M, Stoccoro A, Coppedè F, Furling D, and Simili M
- Subjects
- Biopsy, Cell Differentiation, Cells, Cultured, Endoplasmic Reticulum pathology, Gene Silencing, Humans, Interferon Regulatory Factor-7 genetics, Interferon Type I genetics, MEF2 Transcription Factors metabolism, MicroRNAs metabolism, Microscopy, Electron, Muscle, Skeletal metabolism, MyoD Protein metabolism, Toll-Like Receptor 3 genetics, Autophagy, Interferon Type I metabolism, Muscle Development, Myoblasts metabolism, Myotonic Dystrophy pathology
- Abstract
Congenital myotonic dystrophy type 1 (CDM1) is characterized by severe symptoms that affect patients from birth, with 40% mortality in the neonatal period and impaired skeletal muscle development. In this paper, we examined the relationship between autophagy and abnormal myogenic differentiation of CDM1 myoblasts. We investigated these pathological features at both ultrastructural and molecular levels, utilizing two CDM1 foetal myoblasts, CDM13 and CDM15, with 1800 and 3200 repeats, respectively. The congenital nature of these CDM1 myoblasts was confirmed by the high methylation level at the DMPK locus. Our results indicated that abnormal autophagy was independent of myogenic differentiation, as CDM13 myoblasts differentiated as well as control myoblasts but underwent autophagy like CDM15, displaying impaired differentiation. miRNA expression profiles revealed that CDM15 myoblasts failed to upregulate the complex network of myo-miRNAs under MYOD and MEF2A control, while this network was upregulated in CDM13 myoblasts. Interestingly, the abnormal differentiation of CDM15 myoblasts was associated with cellular stress accompanied by the induction of the interferon type 1 pathway (innate immune response). Indeed, inhibition of the interferon (IFN) type I pathway restores myogenic differentiation of CDM15 myoblasts, suggesting that the inappropriate activation of the innate immune response might contribute to impaired myogenic differentiation and severe muscle symptoms observed in some CDM1 patients. These findings open up the possibility of new therapeutic approaches to treat CDM1.
- Published
- 2018
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34. Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.
- Author
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Dastidar S, Ardui S, Singh K, Majumdar D, Nair N, Fu Y, Reyon D, Samara E, Gerli MFM, Klein AF, De Schrijver W, Tipanee J, Seneca S, Tulalamba W, Wang H, Chai YC, In't Veld P, Furling D, Tedesco FS, Vermeesch JR, Joung JK, Chuah MK, and VandenDriessche T
- Subjects
- Cells, Cultured, Child, Female, Humans, Middle Aged, Muscle Development genetics, Myotonic Dystrophy metabolism, Myotonic Dystrophy pathology, CRISPR-Cas Systems, Gene Editing methods, Induced Pluripotent Stem Cells metabolism, Myoblasts metabolism, Myotonic Dystrophy genetics, Trinucleotide Repeat Expansion genetics
- Abstract
CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.
- Published
- 2018
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35. High Risk of Fatal and Nonfatal Venous Thromboembolism in Myotonic Dystrophy.
- Author
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Sochala M, Porcher R, Stojkovic T, Bécane HM, Béhin A, Laforêt P, Bassez G, Leonard-Louis S, Eymard B, Furling D, Duboc D, and Wahbi K
- Subjects
- Adult, Female, Humans, Incidence, Male, Middle Aged, Myotonic Dystrophy diagnosis, Myotonic Dystrophy mortality, Paris epidemiology, Prevalence, Prognosis, Retrospective Studies, Risk Assessment, Risk Factors, Time Factors, Venous Thromboembolism diagnosis, Venous Thromboembolism mortality, Myotonic Dystrophy epidemiology, Venous Thromboembolism epidemiology
- Published
- 2018
- Full Text
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36. Cells of Matter- In Vitro Models for Myotonic Dystrophy.
- Author
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Matloka M, Klein AF, Rau F, and Furling D
- Abstract
Myotonic dystrophy type 1 (DM1 also known as Steinert disease) is a multisystemic disorder mainly characterized by myotonia, progressive muscle weakness and wasting, cognitive impairments, and cardiac defects. This autosomal dominant disease is caused by the expression of nuclear retained RNAs containing pathologic expanded CUG repeats that alter the function of RNA-binding proteins in a tissue-specific manner, leading ultimately to neuromuscular dysfunction and clinical symptoms. Although considerable knowledge has been gathered on myotonic dystrophy since its first description, the development of novel relevant disease models remains of high importance to investigate pathophysiologic mechanisms and to assess new therapeutic approaches. In addition to animal models, in vitro cell cultures provide a unique resource for both fundamental and translational research. This review discusses how cellular models broke ground to decipher molecular basis of DM1 and describes currently available cell models, ranging from exogenous expression of the CTG tracts to variable patients' derived cells.
- Published
- 2018
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37. rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences.
- Author
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Sellier C, Cerro-Herreros E, Blatter M, Freyermuth F, Gaucherot A, Ruffenach F, Sarkar P, Puymirat J, Udd B, Day JW, Meola G, Bassez G, Fujimura H, Takahashi MP, Schoser B, Furling D, Artero R, Allain FHT, Llamusi B, and Charlet-Berguerand N
- Subjects
- Animals, Binding Sites, Binding, Competitive, Crystallography, X-Ray, Disease Models, Animal, Drosophila melanogaster genetics, Drosophila melanogaster metabolism, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Humans, Kinetics, Models, Molecular, Muscle, Skeletal pathology, Myotonic Dystrophy classification, Myotonic Dystrophy metabolism, Myotonic Dystrophy pathology, Nucleotide Motifs, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, RNA genetics, RNA metabolism, RNA Splicing Factors genetics, RNA Splicing Factors metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Thermodynamics, Muscle, Skeletal metabolism, Myotonic Dystrophy genetics, RNA chemistry, RNA Splicing Factors chemistry, RNA-Binding Proteins chemistry
- Abstract
Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.
- Published
- 2018
- Full Text
- View/download PDF
38. Development and Validation of a New Scoring System to Predict Survival in Patients With Myotonic Dystrophy Type 1.
- Author
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Wahbi K, Porcher R, Laforêt P, Fayssoil A, Bécane HM, Lazarus A, Sochala M, Stojkovic T, Béhin A, Leonard-Louis S, Arnaud P, Furling D, Probst V, Babuty D, Pellieux S, Clementy N, Bassez G, Péréon Y, Eymard B, and Duboc D
- Subjects
- Adult, Cause of Death, Cohort Studies, Female, France, Humans, Life Expectancy, Male, Middle Aged, Myotonic Dystrophy physiopathology, Predictive Value of Tests, Proportional Hazards Models, Regression Analysis, Vital Signs, Myotonic Dystrophy diagnosis, Myotonic Dystrophy mortality
- Abstract
Importance: Life expectancy is greatly shortened in patients presenting with myotonic dystrophy type 1 (DM1), the most common neuromuscular disease. A reliable prediction of survival in patients with DM1 is critically important to plan personalized health supervision., Objective: To develop and validate a prognostic score to predict 10-year survival in patients with DM1., Design, Setting, and Participants: In this longitudinal cohort study, between January 2000 and November 2014, we enrolled 1296 adults referred to 4 tertiary neuromuscular centers in France for management of genetically proven DM1, including 1066 patients in the derivation cohort and 230 in the validation cohort. Data were analyzed from December 2016 to March 2017., Main Outcomes and Measures: Factors associated with survival by multiple variable Cox modeling, including 95% confidence intervals, and development of a predictive score validated internally and externally. Mean values are reported with their standard deviations., Results: Of the 1296 included patients, 670 (51.7%) were women, and the mean (SD) age was 39.8 (13.7) years. Among the 1066 patients (82.3%) in the derivation cohort, 241 (22.6%) died over a median (interquartile range) follow-up of 11.7 (7.7-14.3) years. Age, diabetes, need for support when walking, heart rate, systolic blood pressure, first-degree atrioventricular block, bundle-branch block, and lung vital capacity were associated with death. Simplified score points were attributed to each predictor, and adding these points yielded scores between 0 and 20, with 0 indicating the lowest and 20 the highest risk of death. The 10-year survival rate was 96.6% (95% CI, 94.4-98.9) in the group with 0 to 4 points, 92.2% (95% CI, 88.8-95.6) in the group with 5 to 7 points, 80.7% (95% CI, 75.4-86.1) in the group with 8 to 10 points, 57.9% (95% CI, 49.2-66.6) in the group with 11 to 13 points, and 19.4% (95% CI, 8.6-30.1) in the group with 14 points or more. In 230 patients (17.7%) included in the validation cohort, the 10-year survival rates for the groups with 0 to 4, 5 to 7, 8 to 10, 11 to 13, and 14 points or more were 99.3% (95% CI, 95.0-100), 80.6% (95% CI, 67.1-96.7), 79.3% (95% CI, 66.2-95.1), 43.2% (95% CI, 28.2-66.1), and 21.6% (95% CI, 10.0-46.8), respectively. The calibration curves did not deviate from the reference line. The C index was 0.753 (95% CI, 0.722-0.785) in the derivation cohort and 0.806 (95% CI, 0.758-0.855) in the validation cohort., Conclusions and Relevance: The DM1 prognostic score is associated with long-term survival.
- Published
- 2018
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39. Downregulation of the Glial GLT1 Glutamate Transporter and Purkinje Cell Dysfunction in a Mouse Model of Myotonic Dystrophy.
- Author
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Sicot G, Servais L, Dinca DM, Leroy A, Prigogine C, Medja F, Braz SO, Huguet-Lachon A, Chhuon C, Nicole A, Gueriba N, Oliveira R, Dan B, Furling D, Swanson MS, Guerrera IC, Cheron G, Gourdon G, and Gomes-Pereira M
- Subjects
- Animals, Disease Models, Animal, Down-Regulation, Humans, Mice, Mice, Transgenic, Excitatory Amino Acid Transporter 2 metabolism, Glutamate Plasma Membrane Transport Proteins metabolism, Myotonic Dystrophy metabolism, Purkinje Cells metabolism
- Abstract
Brain function is compromised in myotonic dystrophy type 1 (DM1), but the underlying mechanisms are not fully understood. To gain insight into the cellular and molecular pathways primarily affected, we studied a mouse model of DM1 and brains of adult patients. We found pronounced RNA toxicity in the Bergmann glia of the cerebellum, in association with abnormal Purkinje cell firing and fine motor incoordination in DM1 mice. A global proteomics approach revealed downregulation of the GLT1 glutamate transporter in DM1 mice and human patients, which we found to be the result of MBNL1 inactivation. GLT1 downregulation in DM1 astrocytes increases glutamate neurotoxicity and is detrimental to neurons. Finally, we demonstrated that the upregulation of GLT1 corrected Purkinje cell firing and motor incoordination in DM1 mice. Our findings show that glial defects are critical in DM1 brain pathophysiology and open promising therapeutic perspectives through the modulation of glutamate levels., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
40. Association Between Mutation Size and Cardiac Involvement in Myotonic Dystrophy Type 1: An Analysis of the DM1-Heart Registry.
- Author
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Chong-Nguyen C, Wahbi K, Algalarrondo V, Bécane HM, Radvanyi-Hoffman H, Arnaud P, Furling D, Lazarus A, Bassez G, Béhin A, Fayssoil A, Laforêt P, Stojkovic T, Eymard B, and Duboc D
- Subjects
- 3' Untranslated Regions, Adult, Age Factors, Death, Sudden, Cardiac epidemiology, Death, Sudden, Cardiac pathology, Electrocardiography, Female, Follow-Up Studies, Humans, Incidence, Kaplan-Meier Estimate, Male, Middle Aged, Myotonic Dystrophy genetics, Myotonic Dystrophy mortality, Myotonin-Protein Kinase genetics, Odds Ratio, Pacemaker, Artificial, Phenotype, Prognosis, Regression Analysis, Retrospective Studies, Severity of Illness Index, Sex Factors, Trinucleotide Repeat Expansion genetics, Ventricular Dysfunction, Left diagnosis, Ventricular Dysfunction, Left epidemiology, Myotonic Dystrophy pathology, Registries
- Abstract
Background: In myotonic dystrophy type 1, the association between mutation size (CTG expansion) and the severity of cardiac involvement is controversial., Methods and Results: We selected 855 patients with myotonic dystrophy type 1 (women, 51%; median age, 37 years), with genetic testing performed at the moment of their initial cardiac evaluation, out of 1014 patients included in the Myotonic Dystrophy Type 1-Heart Registry between January 2000 and December 2015. We studied the association between CTG expansion size and other baseline characteristics and (1) cardiac involvement at baseline and (2) the incidence of death, sudden death, and other cardiac adverse events. At initial presentation, the median CTG expansion size was 530 (interquartile range, 300-830). In multivariate analysis, larger expansions were associated with the presence at baseline of conduction defects on the ECG and left ventricular systolic dysfunction. In a median 11.5 years of follow-up period, 210 patients died (25%), including 32 suddenly (4%). Supraventricular arrhythmias developed over lifetime in 166 patients (19%), sustained ventricular tachyarrhythmias in 17 (2%), and permanent pacemakers were implanted in 181 (21%). In Cox regression analyses, larger CTG expansions were significantly associated with (1) total death, sudden death, and pacemaker implantation in a model, including CTG expansion size, age, sex, diabetes mellitus, and (2) all end points except sudden death in a model including all baseline characteristics., Conclusions: The size of the CTG expansion in the blood of myotonic dystrophy type 1 patients is associated with total and sudden deaths, conduction defects, left ventricular dysfunction, and supraventricular arrhythmias., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique Identifier: NCT01136330., (© 2017 American Heart Association, Inc.)
- Published
- 2017
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41. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.
- Author
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Arandel L, Polay Espinoza M, Matloka M, Bazinet A, De Dea Diniz D, Naouar N, Rau F, Jollet A, Edom-Vovard F, Mamchaoui K, Tarnopolsky M, Puymirat J, Battail C, Boland A, Deleuze JF, Mouly V, Klein AF, and Furling D
- Subjects
- Adult, Alternative Splicing drug effects, Alternative Splicing genetics, Cell Line, Transformed, Child, Female, Fibroblasts drug effects, Fibroblasts metabolism, Fibroblasts pathology, Humans, Male, Middle Aged, Muscle Fibers, Skeletal drug effects, Muscle Fibers, Skeletal metabolism, Muscle Fibers, Skeletal pathology, MyoD Protein metabolism, Oligonucleotides, Antisense pharmacology, Oligonucleotides, Antisense therapeutic use, RNA metabolism, Drug Evaluation, Preclinical, Muscle, Skeletal pathology, Myotonic Dystrophy drug therapy, Myotonic Dystrophy pathology
- Abstract
Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations., (© 2017. Published by The Company of Biologists Ltd.)
- Published
- 2017
- Full Text
- View/download PDF
42. Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.
- Author
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Brockhoff M, Rion N, Chojnowska K, Wiktorowicz T, Eickhorst C, Erne B, Frank S, Angelini C, Furling D, Rüegg MA, Sinnreich M, and Castets P
- Subjects
- AMP-Activated Protein Kinases genetics, Adult, Aminoimidazole Carboxamide pharmacology, Animals, Disease Models, Animal, Female, Humans, Male, Mechanistic Target of Rapamycin Complex 1, Mice, Mice, Mutant Strains, Middle Aged, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Muscle Relaxation drug effects, Muscle Relaxation genetics, Myotonic Dystrophy genetics, Myotonic Dystrophy physiopathology, Myotonin-Protein Kinase genetics, Myotonin-Protein Kinase metabolism, Signal Transduction genetics, Sirolimus pharmacokinetics, TOR Serine-Threonine Kinases genetics, TOR Serine-Threonine Kinases metabolism, AMP-Activated Protein Kinases metabolism, Aminoimidazole Carboxamide analogs & derivatives, Multiprotein Complexes antagonists & inhibitors, Muscle Fibers, Skeletal enzymology, Myotonic Dystrophy drug therapy, Myotonic Dystrophy enzymology, Ribonucleotides pharmacology, Signal Transduction drug effects, Sirolimus pharmacology, TOR Serine-Threonine Kinases antagonists & inhibitors
- Abstract
Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease., Competing Interests: M. Sinnreich owns shares of Novartis and is coinventor on a patent application for drug discovery in DM1 (EP 16/166212.7). M. Sinnreich’s institution (University Hospital Basel) has received research support from CSL Behring and Roche, not in relation to this study. C. Angelini is part of the European Board of Genzyme-Sanofi.
- Published
- 2017
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43. Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models.
- Author
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Stantzou A, Ueberschlag-Pitiot V, Thomasson R, Furling D, Bonnieu A, Amthor H, and Ferry A
- Subjects
- Animals, Animals, Newborn, Disease Models, Animal, Female, Male, Mice, Mice, Knockout, Muscular Diseases genetics, Myostatin genetics, Sex Factors, Muscle Contraction genetics, Muscle Strength genetics, Muscle, Skeletal physiology, Muscular Diseases pathology, Myostatin deficiency
- Abstract
Introduction: The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized., Methods: We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KO
Lee ) and (ii) the Grobet model (KOGrobet ), and measured the contraction of tibialis anterior muscle in situ., Results: Absolute maximal isometric force was increased in 6-month-old KOLee and KOGrobet mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KOLee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KOGrobet mice, whereas specific maximal power was reduced only in male KOLee mice., Conclusions: Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017., (© 2016 Wiley Periodicals, Inc.)- Published
- 2017
- Full Text
- View/download PDF
44. Precise small-molecule recognition of a toxic CUG RNA repeat expansion.
- Author
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Rzuczek SG, Colgan LA, Nakai Y, Cameron MD, Furling D, Yasuda R, and Disney MD
- Subjects
- Cells, Cultured, Dose-Response Relationship, Drug, Humans, Molecular Structure, RNA genetics, RNA Splicing drug effects, Small Molecule Libraries chemical synthesis, Structure-Activity Relationship, RNA chemistry, RNA drug effects, Small Molecule Libraries chemistry, Small Molecule Libraries pharmacology, Trinucleotide Repeat Expansion drug effects
- Abstract
Excluding the ribosome and riboswitches, developing small molecules that selectively target RNA is a longstanding problem in chemical biology. A typical cellular RNA is difficult to target because it has little tertiary, but abundant secondary structure. We designed allele-selective compounds that target such an RNA, the toxic noncoding repeat expansion (r(CUG)
exp ) that causes myotonic dystrophy type 1 (DM1). We developed several strategies to generate allele-selective small molecules, including non-covalent binding, covalent binding, cleavage and on-site probe synthesis. Covalent binding and cleavage enabled target profiling in cells derived from individuals with DM1, showing precise recognition of r(CUG)exp . In the on-site probe synthesis approach, small molecules bound adjacent sites in r(CUG)exp and reacted to afford picomolar inhibitors via a proximity-based click reaction only in DM1-affected cells. We expanded this approach to image r(CUG)exp in its natural context., Competing Interests: The authors declare no competing financial interests.- Published
- 2017
- Full Text
- View/download PDF
45. CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.
- Author
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van Agtmaal EL, André LM, Willemse M, Cumming SA, van Kessel IDG, van den Broek WJAA, Gourdon G, Furling D, Mouly V, Monckton DG, Wansink DG, and Wieringa B
- Subjects
- Animals, Bacterial Proteins genetics, Base Sequence, CRISPR-Associated Protein 9, Clustered Regularly Interspaced Short Palindromic Repeats, Codon, Disease Models, Animal, Endonucleases genetics, Fibroblasts metabolism, Gene Expression, Gene Order, Genetic Loci, Humans, Mice, RNA, Guide, CRISPR-Cas Systems, RNA, Messenger genetics, RNA, Messenger metabolism, Sequence Deletion, CRISPR-Cas Systems, Gene Editing, Genomic Instability, Myotonic Dystrophy genetics, Myotonin-Protein Kinase genetics, Trinucleotide Repeat Expansion, Trinucleotide Repeats
- Abstract
Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)
n -repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
46. Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins.
- Author
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Alves S, Marais T, Biferi MG, Furling D, Marinello M, El Hachimi K, Cartier N, Ruberg M, Stevanin G, Brice A, Barkats M, and Sittler A
- Subjects
- Animals, Ataxin-7 genetics, DNA-Binding Proteins genetics, Disease Models, Animal, Female, Humans, Lentivirus genetics, Mice, Inbred C57BL, Neurons metabolism, Phenotype, Ataxin-7 metabolism, DNA-Binding Proteins metabolism, RNA-Binding Proteins metabolism, Spinocerebellar Ataxias genetics
- Abstract
Background: We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure., Results: LV-mediated overexpression of MUT ATXN7 into the cerebellum of C57/BL6 adult mice induced neuropathological features similar to that observed in patients, such as intranuclear aggregates in Purkinje cells (PC), loss of synaptic markers, neuroinflammation, and neuronal death. No neuropathological changes were observed when truncated wild-type ataxin-7 (WT ATXN7) was injected. Interestingly, the local delivery of LV-expressing mutant ataxin-7 (LV-MUT-ATXN7) into the cerebellum of wild-type mice also mediated the development of an ataxic phenotype at 8 to 12 weeks post-injection. Importantly, our data revealed abnormal levels of the FUS/TLS, MBNL1, and TDP-43 RNA-binding proteins in the cerebellum of the LV-MUT-ATXN7 injected mice. MUT ATXN7 overexpression induced an increase in the levels of the pathological phosphorylated TDP-43, and a decrease in the levels of soluble FUS/TLS, with both proteins accumulating within ATXN7-positive intranuclear inclusions. MBNL1 also co-aggregated with MUT ATXN7 in most PC nuclear inclusions. Interestingly, no MBNL2 aggregation was observed in cerebellar MUT ATXN7 aggregates. Immunohistochemical studies in postmortem tissue from SCA7 patients and SCA7 knock-in mice confirmed SCA7-induced nuclear accumulation of FUS/TLS and MBNL1, strongly suggesting that these proteins play a physiopathological role in SCA7., Conclusions: This study validates a novel SCA7 mouse model based on lentiviral vectors, in which strong and sustained expression of MUT ATXN7 in the cerebellum was found sufficient to generate motor defects.
- Published
- 2016
- Full Text
- View/download PDF
47. Dystrophin restoration therapy improves both the reduced excitability and the force drop induced by lengthening contractions in dystrophic mdx skeletal muscle.
- Author
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Roy P, Rau F, Ochala J, Messéant J, Fraysse B, Lainé J, Agbulut O, Butler-Browne G, Furling D, and Ferry A
- Subjects
- Action Potentials, Animals, Dependovirus genetics, Disease Models, Animal, Dystrophin genetics, Genetic Predisposition to Disease, Genetic Vectors, Mice, Inbred mdx, Muscle Strength, Muscle, Skeletal physiopathology, Muscular Dystrophy, Duchenne genetics, Muscular Dystrophy, Duchenne metabolism, Muscular Dystrophy, Duchenne physiopathology, Phenotype, RNA, Small Nuclear metabolism, Time Factors, Up-Regulation, Dystrophin metabolism, Excitation Contraction Coupling, Genetic Therapy, Muscle, Skeletal metabolism, Muscular Dystrophy, Duchenne therapy, RNA, Small Nuclear genetics
- Abstract
Background: The greater susceptibility to contraction-induced skeletal muscle injury (fragility) is an important dystrophic feature and tool for testing preclinic dystrophin-based therapies for Duchenne muscular dystrophy. However, how these therapies reduce the muscle fragility is not clear., Methods: To address this question, we first determined the event(s) of the excitation-contraction cycle which is/are altered following lengthening (eccentric) contractions in the mdx muscle., Results: We found that the immediate force drop following lengthening contractions, a widely used measure of muscle fragility, was associated with reduced muscle excitability. Moreover, the force drop can be mimicked by an experimental reduction in muscle excitation of uninjured muscle. Furthermore, the force drop was not related to major neuromuscular transmission failure, excitation-contraction uncoupling, and myofibrillar impairment. Secondly, and importantly, the re-expression of functional truncated dystrophin in the muscle of mdx mice using an exon skipping strategy partially prevented the reductions in both force drop and muscle excitability following lengthening contractions., Conclusion: We demonstrated for the first time that (i) the increased susceptibility to contraction-induced muscle injury in mdx mice is mainly attributable to reduced muscle excitability; (ii) dystrophin-based therapy improves fragility of the dystrophic skeletal muscle by preventing reduction in muscle excitability.
- Published
- 2016
- Full Text
- View/download PDF
48. Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy.
- Author
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Freyermuth F, Rau F, Kokunai Y, Linke T, Sellier C, Nakamori M, Kino Y, Arandel L, Jollet A, Thibault C, Philipps M, Vicaire S, Jost B, Udd B, Day JW, Duboc D, Wahbi K, Matsumura T, Fujimura H, Mochizuki H, Deryckere F, Kimura T, Nukina N, Ishiura S, Lacroix V, Campan-Fournier A, Navratil V, Chautard E, Auboeuf D, Horie M, Imoto K, Lee KY, Swanson MS, de Munain AL, Inada S, Itoh H, Nakazawa K, Ashihara T, Wang E, Zimmer T, Furling D, Takahashi MP, and Charlet-Berguerand N
- Subjects
- Adult, Aged, Animals, Base Sequence, Binding Sites, Computer Simulation, Electrophysiological Phenomena, Exons genetics, Female, HEK293 Cells, Heart Conduction System pathology, Humans, Male, Middle Aged, Molecular Sequence Data, NAV1.5 Voltage-Gated Sodium Channel metabolism, Nucleotide Motifs genetics, RNA-Binding Proteins metabolism, Sodium Channels metabolism, Xenopus, Alternative Splicing genetics, Arrhythmias, Cardiac complications, Arrhythmias, Cardiac genetics, Heart Conduction System physiopathology, Myotonic Dystrophy complications, Myotonic Dystrophy genetics, NAV1.5 Voltage-Gated Sodium Channel genetics
- Abstract
Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.
- Published
- 2016
- Full Text
- View/download PDF
49. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin.
- Author
-
Laustriat D, Gide J, Barrault L, Chautard E, Benoit C, Auboeuf D, Boland A, Battail C, Artiguenave F, Deleuze JF, Bénit P, Rustin P, Franc S, Charpentier G, Furling D, Bassez G, Nissan X, Martinat C, Peschanski M, and Baghdoyan S
- Abstract
Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.
- Published
- 2015
- Full Text
- View/download PDF
50. MyoD transcription factor induces myogenesis by inhibiting Twist-1 through miR-206.
- Author
-
Koutalianos D, Koutsoulidou A, Mastroyiannopoulos NP, Furling D, and Phylactou LA
- Subjects
- Cell Differentiation genetics, Cell Differentiation physiology, Chromatin Immunoprecipitation, Humans, Muscle Development genetics, Muscle Development physiology, MyoD Protein genetics, Myoblasts cytology, Myoblasts metabolism, MicroRNAs genetics, MyoD Protein metabolism
- Abstract
Twist-1 is mostly expressed during development and has been previously shown to control myogenesis. Because its regulation in muscle has not been fully exploited, the aim of this project was to identify micro (mi)RNAs in muscle that regulate Twist-1. miR-206, one of the most important muscle-specific miRNAs (myomiRs), was identified as a possible regulator of Twist-1 mRNA. Luciferase assays and transfections in human foetal myoblasts showed that Twist-1 is a direct target of miR-206 and that through this pathway muscle cell differentiation is promoted. We next investigated whether MyoD, a major myogenic transcription factor, regulates Twist-1 because it is known that MyoD induces expression of the miR-206 gene. We found that forced MyoD expression induced miR-206 upregulation and Twist-1 downregulation through binding to the miR-206 promoter, followed by increased muscle cell differentiation. Finally, experiments were performed in muscle cells from subjects with congenital myotonic dystrophy type 1, in which myoblasts fail to differentiate into myotubes. MyoD overexpression inhibited Twist-1 through miR-206 induction, which was followed by an increase in muscle cell differentiation. These results reveal a previously unidentified mechanism of myogenesis that might also play an important role in muscle disease., (© 2015. Published by The Company of Biologists Ltd.)
- Published
- 2015
- Full Text
- View/download PDF
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